Farnesyl pyrophoshate synthase protein, nucleic acid and promoter region thereof

ABSTRACT

The promoter region of the farnesyl pyrophosphate synthase gene that was expressed in the hop luplin gland in a specific manner was elucidated based on the genomic DNA of the hop farnesyl pyrophosphate synthase gene having the nucleotide sequence set forth in SEQ ID NO:2, the cDNA of the hop farnesyl pyrophosphate synthase gene having the nucleotide sequence set forth in SEQ ID NO:3, and the nucleotide sequence information on the genomic DNA and the cDNA. It will reveal the gene involved in the biosynthesis of secondary metabolites in a hop as well as the nucleotide sequence of the promoter gene that functions in the hop luplin gland in a tissue-specific manner. This will allow for the transformation of the hop by gene manipulations and the in vitro synthesis of the hop secondary metabolites.

TECHNICAL FIELD

[0001] This invention relates to farnesyl pyrophosphate (FPP) synthasegenes of hop and their promoter regions.

BACKGROUND ART

[0002] Plants produce and accumulate within their bodies, numerous kindsof low molecular weight organic compounds such as terpenoids, alkaloids,phenolics, and saponins. It was initially thought that these compoundswere not directly responsible for the life maintenance of organisms andhave only auguxilliary functions; therefore, they were referred to as“secondary metabolites.”

[0003] However, in recent years it has been beginning to be understoodthat these secondary metabolites function as substances responsible forthe cell differentiation or the defense against exogenous factors.Concurrently, the methods of utilization are being found in the broadfields of taste products, drugs and pigments. Not to mention theagricultural field, their utility is catching attention in broad fields.

[0004] Because such secondary metabolites are valuable in beingindustrially utilized, the elucidation of their formation process inplant cells has progressed and presently it has been shown that thesubstances are synthesized through a complex cascade involving a largenumber of enzymes. Direct Extraction from plants is, however, needed toobtain such substances. In those cases the quantities that can beisolated at one time are very small, resulting in high cost. Therefore,it has been desired that in vitro synthetic methods using genemanipulations or cultured cells be developed.

[0005] Farnesyl pyrophosphate synthase is known as an enzyme that isinvolved in the synthetic cascade of the secondary metabolites inplants. Farnesyl pyrophosphate synthase is an enzyme that is involved inthe metabolism of isoprenoids which forms the basis for a variety ofsubstances in plants such as pigments, odorants, phytohormones,phytoalexins, and defense substances against pests (Plant Biochemistry &Molecular Biology, Hans-Walter Heldt, Oxford University Press, pp.360-376, 1997). It has been shown that farnesyl pyrophosphate synthasecatalyzes the reaction converting isopentenyl pyrophosphate into geranylpyrophosphate by adding dimethylallyl pyrophosphate thereto as well ascatalyzes the reaction converting said geranyl pyrophosphate intofarnesyl pyrophosphate by adding isopentenyl pyrophosphate thereto.

[0006] Hop is a principal material to give beer refreshing bitter tasteand flavor. It is beginning to be understood that the secondarymetabolites are secreted in large quantities in luplin gland containedin the cone of the hop and these secondary metabolites greatlycontribute to the bitter taste and flavor of beer. Further, in recentyears it has been shown that the secondary metabolites of the hoppossess pharmacological actions (for example, Biosci. Biotech. Biochem.,61 (1), 158, 1997). Under such circumstances, a variety of breedingimprovements are being made to the hop with an emphasis on the secondarymetabolites accumulated in the luplin gland such as bitter substancesand essential oil constituents.

[0007] However, the hop is a dioecious plant. Especially, the male plantdoes not beer cones that serves as the beer material and thus is notregarded important commercially. Very little study, therefore, has beencarried out and the genetic traits that will be useful for fermentationhave been hardly elucidated. For these reasons, conventional breedingmethods through crossing largely rely on experience and intuition. Thepresent situation is that the brewing qualities are totallyunpredictable until the cones have grown. Accordingly, it is stronglydesired that the farnesyl pyrophosphate synthase gene be isolated from ahop and that the control of the secondary metabolites in the hop and invitro synthetic methods be established according to an approach usinggene manipulations.

[0008] The breeding methods utilizing genetic engineering such astransformation techniques and molecular selection techniques arebecoming possibilities in various plants. In these methods, moreobjective and efficient breeding is possible as compared to theconventional breeding methods which largely rely on experience andintuition. More specifically, the transformation technique is one inwhich an exogenous gene is introduced into a plant cell to have itexpressed and the trait to be desirably incorporated is directlyintroduced into the cell. In order to have the exogenous gene expressed,the following method may be employed: an objective structural gene and aterminator operable in a plant cell are linked to an operable promotercapable of regulating the expression of the gene in the plant cell andthe linkage is introduced into the plant cell. For a promoter that isfrequently used at the experimental level, there are mentioned, amongothers, a CaMV 35S promoter, a nopaline synthase gene promoter both ofwhich can express transgenes in relatively large kinds of plantsregardless of their tissues (Sanders P. R. et al., Nucleic Acid Res, 15(1987) 1543-1558). However, when the aforementioned promoters are usedto express the transgenes in all the tissues, some transgenes may doharm to the growth of the plants. It is, therefore, strongly desiredthat tissue-specific promoters capable of expressing exogenous genes inthe objective tissue be isolated.

DISCLOSURE OF THE INVENTION

[0009] This invention has been made in view of the problems that areinherent in the aforementioned prior art; it aims at elucidating thegenes involved in the biosynthesis of secondary metabolites in a hop andthe nucleotide sequences (base sequences) of the promoters operable in atissue-specific manner in the luplin gland of the hop as well as aims atallowing for the transformation of the hop by gene manipulations and thein vitro synthesis of the secondary metabolite of the hop.

[0010] As a result of having pursued diligent investigations toaccomplish the above-stated objects, the present inventors foundfarnesyl pyrophosphate synthase genes and their promoter genes, whichled to the completion of this invention: the farnesyl pyrophosphatesynthase genes were strongly expressed in the luplin gland of the hopand were involved in the biosynthesis of secondary metabolites.

[0011] Specifically, according to this invention there are provided theproteins described in 1-2 below:

[0012] 1. A protein having the amino acid sequence set forth in SEQ IDNO:1 in the Sequence Listing.

[0013] 2. A protein having an amino acid sequence derivable from thedeletion or the substitution of one or more amino acids in the aminoacid sequence set forth in SEQ ID NO:1 in the Sequence Listing, or fromthe addition of one or more amino acids to the amino acid sequence setforth in SEQ ID NO:1 in the Sequence Listing, said protein possessingthe farnesyl pyrophosphate synthase activity.

[0014] Also, according to this invention there are provided the nucleicacids described in 3-10 below:

[0015] 3. A nucleic acid encoding a protein having the amino acidsequence set forth in SEQ ID NO:1 in the Sequence Listing.

[0016] 4. A nucleic acid having the nucleotide sequence set forth in SEQID NO:3 in the Sequence Listing.

[0017] 5. A nucleic acid comprising a part of the nucleotide sequenceset forth in SEQ ID NO:3 in the Sequence Listing.

[0018] 6. A nucleic acid that hybridizes to a nucleic acid having thenucleotide sequence set forth in SEQ ID NO:3 in the Sequence Listing orto a complementary nucleic acid thereof under stringent conditions, saidnucleic acid encoding a protein possessing the farnesyl pyrophosphatesynthase activity.

[0019] 7. A nucleic acid having the nucleotide sequence set forth in SEQID NO:2 in the Sequence Listing.

[0020] 8. A nucleic acid comprising a part of the nucleotide sequenceset forth in SEQ ID NO:2 in the Sequence Listing.

[0021] 9. A nucleic acid having a nucleotide sequence of from base No. 1to base No. 1886 in the nucleotide sequence set forth in SEQ ID NO:2 inthe Sequence Listing.

[0022] 10. A nucleic acid that hybridizes to a nucleic acid having thenucleotide sequence described in 7 or 8 above or to a complementarynucleic acid thereof under stringent conditions, said nucleic acidpossessing promoter activity.

[0023] The use of these proteins or nucleic acids will then enable thetransformation of a hop by gene manipulations as well as enable the invitro synthesis of the secondary metabolites of a hop.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a diagram showing nucleic acid fragments obtained fromthe isolation process of a farnesyl pyrophosphate synthase gene of thisinvention.

[0025]FIG. 2 is a schematic diagram showing the principle of Inverse PCRused in the invention.

[0026]FIG. 3 is a schematic diagram showing the principle ofCasette-ligation mediate PCR used in the invention.

[0027]FIG. 4 is a representation showing the developed image of thinlayer chromatography used when the activity of the farnesylpyrophosphate synthase of the invention was determined.

[0028]FIG. 5 is a photograph of Northern analysis that confirmed theexpression of the farnesyl pyrophosphate synthase gene of the invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] The preferred embodiments of this invention will be described indetail hereafter by referring to the drawings when necessary.

[0030] As used in the invention, “nucleic acid” refers to DNA, RNA, or apolynucleotide that may be a derivatized active DNA or RNA among others.Preferred are DNA and/or RNA. In this case, the forms of the nucleicacid include genomic DNA, cDNA and mRNA, for example.

[0031] As used in the invention, “hybridize under stringent conditions”means that two nucleic acid fragments hybridize to each other under thehybridization conditions as described in Sambrook, J. et al.,“Expression of Cloned Genes in E. coli,” Molecular Cloning: A LaboratoryManual (1989) Cold Spring Harbor Laboratory Press, New York, USA,9.47-9.62 and 11.45-11.61.

[0032] More specifically, “stringent conditions” means that afterhybridization in 6.0×SSC at about 45° C., washing is to be done in2.0×SSC at 50° C., for example. For the purpose of selection ofstringency, the salt concentration in the washing step can be selectedto be from about 2.0×SSC at 50° C. under low stringency to about 0.1×SSCat 50° C. under high stringency. Furthermore, the temperature of washingstep can be increased from room temperature under low stringencyconditions (about 22° C.) to about 65° C. under high stringencyconditions.

[0033] As used in the invention, “promoter” refers to a nucleotidesequence present in DNA that is a signaling sequence functioning togovern directing the initiation and termination of RNA synthesis(transcription) or directing the regulation of its frequency.

[0034] As used in the invention, “promoter activity” refers to thefunction of initiating, terminating, and regulating transcription by thepromoter as described above.

[0035] The hop farnesyl pyrophosphate synthase of this invention will befirst described.

[0036] The protein of this invention is a hop farnesyl pyrophosphatesynthase protein having the amino acid sequence with 342 amino acidresidues, as set forth in SEQ ID NO:1 in the Sequence Listing.

[0037] Also, the protein of the invention may be a protein having anamino acid sequence derivable from the deletion or the substitution ofone or more amino acids in the amino acid sequence set forth in SEQ IDNO:1 in the Sequence Listing, or from the addition of one or more aminoacids to the amino acid sequence set forth in SEQ ID NO:1 in theSequence Listing, insofar as it possesses the farnesyl pyrophosphatesynthase activity.

[0038] Further, since sugar chains may be appended to a large number ofproteins, the addition of sugar chains can be adjusted by subjecting oneor more amino acids to conversion. Therefore, proteins for which theaddition of sugar chains within the amino acid sequence set forth in SEQID NO:1 in the Sequence Listing has been adjusted are also embraced bythe proteins of this invention insofar as they possess the farnesylpyrophosphate synthase activity as described above.

[0039] Further, nucleic acids having nucleotide sequences encoding theaforementioned farnesyl pyrophosphate synthase proteins are alsoembraced by the invention. Specifically, because a plurality ofnucleotide sequences (or codons) encoding a single amino acid exist,there are a large number of nucleic acids encoding the amino acidsequence set forth in SEQ ID NO:1 in the Sequence Listing. These nucleicacids are thus embraced by the nucleic acids of this invention. As usedherein, “encode a protein” means that when DNA is double-stranded, theterm includes a DNA wherein either of the complementary two strands isprovided with the nucleotide sequence encoding the protein. Therefore,embraced by the invention are nucleic acids comprising the nucleotidesequences directly encoding the amino acid sequence set forth in SEQ IDNO:1 in the Sequence Listing and nucleic acids comprising the nucleotidesequences complementary to the foregoing nucleotide sequences.

[0040] Next, the farnesyl pyrophosphate synthase genes according to theinvention will be described.

[0041] The nucleic acid of this invention is one (cDNA) having thenucleotide sequence set forth in SEQ ID NO:3 in the Sequence Listing andencoding a hop farnesyl pyrophosphate synthase.

[0042] The nucleic acid of the invention may also be a part of thenucleotide sequence set forth in SEQ ID NO:3 in the Sequence Listing.

[0043] Further, the nucleic acid of the invention may be one thathybridizes to the nucleotide sequence with 1029 bases set forth in SEQID NO:3 in the Sequence Listing under stringent conditions and encodinga protein possessing the farnesyl pyrophosphate synthase activity; andits nucleotide sequence is not particularly limited insofar as itsatisfies these conditions. Still further, the nucleic acids of theinvention embrace nucleic acids having nucleotide sequencescomplementary to that of the aforementioned nucleic acid undergoinghybridization under stringent conditions. Specifically, there arementioned, among others, nucleic acids having deletions of,substitutions of, insertions to or additions to several bases of thenucleic acid having the nucleotide sequence of SEQ ID NO:3 andpossessing the farnesyl pyrophosphate synthase activity. As used herein,“deletion, substitution, insertion, or addition” includes not only ashort deletion, substitution, insertion or addition with 1 to 10 bases,but also a long deletion, substitution, insertion or addition with 10 to100 bases. The “farnesyl pyrophosphate synthase activity” refers to theactivity allowing the reaction to proceed by which farnesylpyrophosphate is synthesized by the catalytic action of the farnesylpyrophosphate synthase. In this case the substance that serves as asubstrate for the farnesyl pyrophosphate synthase is not particularlylimited insofar as it is a substance from which farnesyl pyrophosphatecan be ultimately synthesized. Specifically, there are mentionedisopentenyl pyrophosphate and geranyl pyrophosphate.

[0044] The geranyl pyrophosphate and farnesyl pyrophosphate are regardedin a hop as the precursors of essential oil constituents such asmyrcenes, humulenes, caryophyllenes, and farnesenes. Thus, by detectinga nucleic acid having the nucleotide sequence set forth in SEQ ID NO:3in the Sequence Listing, it will be possible to utilize the nucleic acidas a genetic marker for the traits concerning the regulation of themetabolic system for hop's essential oil constituents and the essentialoil constituents themselves. The detection of a nucleic acid does notrequire the whole nucleotide sequence set forth in SEQ ID NO:3 in theSequence Listing; for example, part of the sequence may be amplified byPCR and the detection may be carried out by gene analysis techniquessuch as nucleotide sequencing (base sequencing) or RFLP (RestrictionFragment Length Polymorphism). Therefore, the nucleic acids of thisinvention embrace nucleic acids comprising a part of the nucleotidesequence set forth in SEQ ID NO:3 in the Sequence Listing.

[0045] Next, the promoter region of the farnesyl pyrophosphate synthasegene according to this invention will be described.

[0046] The nucleic acid of this invention is a nucleic acid having thenucleotide sequence set forth in SEQ ID NO:2 in the Sequence Listing.The nucleic acid of the invention may be part of the nucleic acid havingthe nucleotide sequence with 4699 bases, as set forth in SEQ ID NO:2 inthe Sequence Listing, or alternatively it may be the nucleotide sequencerepresented by base no. 1 to no. 1886.

[0047] The nucleic acid having the nucleotide sequence set forth in SEQID NO:2 is genomic DNA of farnesyl pyrophosphate synthase and thenucleotide sequence encoding the starting methionine of farnesylpyrophosphate synthase is from base no. 1887 to no. 1889 within thegenomic DNA. Thus, base no. 1 to no. 1886 in the nucleotide sequence setforth in SEQ ID NO:2 in the Sequence Listing is a 5′-noncoding region,within which the promoter region of the farnesyl pyrophosphate synthasegene is contained. It will be difficult to unambiguously define theboundaries of both ends of the promoter region within base no. 1 to no.1886; therefore, the nucleic acids of this invention embrace sequencescomprising part of base no. 1 to no. 1886 insofar as they possess thepromoter activity.

[0048] The nucleic acid of this invention may be one that hybridizes tothe nucleic acid having the nucleotide sequence set forth in SEQ ID NO:2or to the nucleic acid having the nucleotide sequence represented bybase no. 1 to no. 1886 within the foregoing nucleic acid under stringentconditions and that possess the promoter activity; and its nucleotidesequence is not particularly limited. The nucleic acids of thisinvention further embrace nucleic acids having the nucleotide sequencecomplementary to that of the nucleic acid that undergoes hybridizationunder stringent conditions and that possesses the promoter activity.Specifically, there are mentioned, among others, nucleic acids havingone or more deletions in, substitutions in, insertions to or addition tothe nucleic acid having the nucleotide sequence of SEQ ID NO:2 andpossessing the promoter activity. As used herein, “deletion,substitution, insertion, or addition” includes not only a shortdeletion, substitution, insertion or addition with 1 to 10 bases, butalso a long deletion, substitution, insertion or addition with 10 to 100bases.

[0049] Next, the preferred method of isolating the nucleic acids of thisinvention and analyzing the functions of their gene products will bedescribed.

[0050] The nucleic acid of the invention can be isolated through steps(1) to (5) as described below and it is possible to confirm in steps (6)to (7) that the isolated gene will display the farnesyl pyrophosphatesynthase activity or will possess the promoter activity.

[0051] 1. Isolation of Farnesyl Pyrophosphate Synthase Gene and itsPromoter from a Hop

[0052] (1) Preparation of Hop Genomic DNA

[0053] The preparation of the hop genomic DNA can be carried outaccording to a method known in the art: for example, the method ofWagner, D. B. et al. (Proc. Natl. Acad. Sci. USA 84, 2097-2100 (1987))can be followed.

[0054] (2) Isolation of Farnesyl Pyrophosphate Synthase Gene and itsPromoter

[0055] Partial fragments of the farnesyl pyrophosphate synthase gene canbe isolated by designing primers based on the known nucleotide sequencesof the farnesyl pyrophosphate synthase genes from other plants such asArabidopsis and corn and by using the known methods such as Inverse PCRor Cassette-ligation mediated PCR. As Inverse PCR is shown in theschematic diagram of FIG. 2, DNA that serves as a sample is digestedwith restriction enzymes; the restriction enzyme-digested product isthen cyclized to a sample that serves as a template prior toamplification; and primers synthesized in directions opposite to thosefor the primers to be used in ordinary PCR are used to perform PCR. Themethod makes it possible to amplify the upstream or downstream regionadjacent to a specific nucleotide sequence. As a concrete example ofInverse PCR, there may be mentioned the method of Liu, Y. G. et al.(Genomics 25, 674-681 (1995)). As schematically shown in FIG. 3,Cassette-ligation Mediated PCR is a method to be used when an unknownnucleotide sequence adjacent to a known nucleotide sequence will bedesirably determined (e.g., the method as described in the protocolattached to a Takara LA PCR in vitro Cloning Kit (Takara Shuzo Co.Ltd.)). Specifically, the nucleic acid containing such nucleic acidregion is first digested with restriction enzyme. Adapter nucleic acidsof known nucleotide sequences having the restriction enzyme recognitionsites from which primers can be designed are ligated to theaforementioned nucleic acid. Then, the unknown nucleotide sequenceregion flanked by the known sequence regions is amplified by PCR and theamplified product may be sequenced. By repeating such Inverse PCR and/orCassette-ligation Mediated PCR, it is possible to isolate the entireregion of the hop farnesyl pyrophosphate synthase gene and its promoter.

[0056] (3) Sequencing

[0057] The isolated genes can be sequenced by a method known in the art:for example, it can be done by following the protocol attached to an ABIPRISM Dye Primer Cycle Sequencing Ready Reaction Kit available from PEBiosystems Inc. The nucleotide sequences determined by theaforementioned method can be subjected to homology search using adatabase such as http://www.ncbi.nlm.nih.gov/BLAST and thus it will bepossible to find out the presence or absence of homology with the knowngenes obtained from other species of plants as well as the degree ofhomology. This will enable determination as to whether or not theobtained genes are novel.

[0058] (4) Preparation of Total RNA from the Respective Tissue Fractions

[0059] After an arbitrary fraction has been prepared, the preparation oftotal RNA can be carried out according to a method known in the art: forexample, the method of Chang, S. et al. (Plant Molecular Biology Report11, 113-116 (1993)) can be followed.

[0060] (5) Isolation of Farnesyl Pyrophosphate Synthase Gene cDNA

[0061] cDNA of the farnesyl pyrophosphate synthase gene can be isolatedaccording to a method known in the art: specifically, primers may bedesigned based on the nucleotide sequence of the genomic DNA for thefarnesyl pyrophosphate synthase gene isolated in (2) and cDNAsynthesized from the total mRNA may be used as a template to effectisolation through RT-PCR. For a concrete method in this case, the methodas described in the protocol attached to a Titan One Tube RT-PCR Systemavailable from Roche Diagnostics Inc. can be employed, for example.

[0062] (6) Functional Analysis of the Protein Encoded by the IsolatedFarnesyl Pyrophosphate Synthase Gene

[0063] The protein encoded by the farnesyl pyrophosphate synthase geneisolated in (5) can be expressed in E. coli cells by incorporating cDNAof the farnesyl pyrophosphate synthase gene into an expression vectorand introducing the vector into an E. coli cell. The expression andpurification of the protein encoded by the farnesyl pyrophosphatesynthase gene described above can, for example, be carried out by themethod as described in the protocol attached to a QIAexpress ExpressionSystem (QIAGEN Inc.). The functions of the farnesyl pyrophosphatesynthase protein expressed in those E. coli cells and purified can beconfirmed by a method known in the art: for example, the method ofSylvie A. et al. (Arch. Biochem. Biophys. 321, 493-500, (1995)) can beused for confirmation.

[0064] (7) Northern Hybridization (Hereunder Referred to as “NorthernAnalysis”)

[0065] The isolated farnesyl pyrophosphate synthase gene can be used asa probe and Northern analysis can be done to analyze as to in whichtissue the isolated farnesyl pyrophosphate synthase gene is expressed,or as to in which tissue the isolated farnesyl pyrophosphate synthasefunctions. For example, the analysis can be done by the method asdescribed in “The DIG System User's Guide for Filter Hybridization”(Boeringer Manheim) p. 53-55 (1995).

[0066] Next, one embodiment that is made possible by the nucleic acid ofthis invention will be described.

[0067] (1) Probes for Use in Hybridization

[0068] By using a part or the whole of the nucleotide sequence disclosedin this invention as a hybridization probe, it is possible to at leastdetect the farnesyl pyrophosphate synthase gene expressed in a hop. Byusing a part or the whole of the nucleotide sequence disclosed in thisinvention as a hybridization probe to investigate the gene expression inhop tissues, it is also possible to identify the distribution of thegene expression.

[0069] When a part or the whole of the nucleotide sequence disclosed inthis invention is used as a hybridization probe, the method ofhybridization itself is not particularly limited; however, there arespecifically mentioned as examples, Northern hybridization, Southernhybridization, colony hybridization, dot hybridization, Fluorescence insitu hybridization (FISH), in situ hybridization (ISH), DNA chip method,and microarray method.

[0070] When the nucleotide sequence of this invention is used as thehybridization probe, the nucleotide length (base length) of at least 20bases is necessary; and a gene having a nucleotide length of 20 or moreconsecutive bases within the gene sequence of the invention ispreferably used. There are used more preferably, one having a nucleotidelength of 40 or more bases, and most preferably one having a nucleotidelength of 60 or more bases.

[0071] The nucleic acid probe technique is well known to one skilled inthe art, and suitable hybridization conditions for the probes withindividual lengths according to this invention and the objectivepolynucleotide can readily be determined. To obtain hybridizationconditions optimal for the probes containing varying lengths, suchmanipulations are well known to one skilled in the art. For example,Sambrooks et al., “Molecular Cloning: A Laboratory Manual,” 2nd. Ed.,Cold Spring Harbor (1989) may be referred to.

[0072] Here, the probes are preferably labeled so that they can beeasily detected. Detectable labels may be any type or a portion thereofthat can be detected either by the naked eyes or with a device. Thedetectable labels that are ordinarily used are, for example, radioactivelabels such as ³²P, ¹⁴C, ¹²⁵I, ³H, and ³⁵S. Biotin-labeled nucleotidescan be incorporated into nucleic acids by using nick translation,chemical and enzymatic means, or the like. The biotin-labeled probes aredetected after hybridization utilizing labeling means such asavidin/streptavidin, fluorescent labels, enzymes, and gold colloidalcomplexes. The nucleic acids may be labeled by being bound to proteins.Alternatively, there may be used nucleic acids that have cross-linked tothe radioactive or fluorescent histone single strand biding protein.

[0073] (2) Primers for Use in PCR

[0074] It is also possible to detect the farnesyl pyrophosphate synthasegene by using as primer any sequence of the disclosed nucleotidesequence and the Polymerase Chain Reaction (PCR) method. For example,RNA can be extracted from a sample to be assayed and the gene expressioncan be semi-quantitatively determined by RT-PCR. Such a method can becarried out by a technique well known in the art.

[0075] When the nucleic acid of this invention is used as a PCR primer,the nucleotide length of from 10 to 60 bases is necessary; and a nucleicacid having a nucleotide length of from 10 to 60 consecutive bases (morepreferably 15 to 30 bases) within the nucleic acid of the invention ispreferably used. Generally, the GC content of the primer sequence ispreferably from 40 to 60%. Further, it is preferred that there be nodifference in Tm value between two primers. It is also preferred thatannealing do not take place at the 3′-ends of the primers and asecondary structure do not occupy within the primers.

[0076] (3) Screening for Nucleic Acids

[0077] It is possible to detect the distribution of expression of thefarnesyl pyrophosphate synthase gene that is expressed in a hop by usinga part or the whole of the nucleotide sequence disclosed in thisinvention. For example, the part or the whole of the nucleotide sequencedisclosed in the invention can be used as a hybridization probe or as aprimer in PCR to detect the distribution of gene expression.

[0078] DNA chips, microarrays or the like can also be used to detectsaid distribution of gene expression. Specifically, parts or the wholeof the nucleotide sequence disclosed in this invention can all beapplied onto the chip or the array directly. RNA extracted from cells islabeled with a fluorescent substance or the like and is hybridized tothe chip or the array; it is then possible to analyze as to in whichcell the gene is highly expressed. DNA applied onto the chip or thearray may be a PCR product obtained by using the part or the whole ofthe nucleotide sequence disclosed in this invention.

[0079] (4) DNA Cloning

[0080] It is possible to clone a gene that is expressed at least in ahop by using a part or the whole of the nucleotide sequence disclosed inthis invention. For example, the part or the whole of the nucleotidesequence disclosed in the invention can be used as a probe in Northernhybridization or in colony hybridization, or as a primer in PCR to clonethe part or the whole of the nucleotide sequence disclosed in thisinvention.

[0081] In embodiments other than those described above, it will bepossible to obtain information on the hop farnesyl pyrophosphatesynthase or to carry out the transformation of a hop and the productionof its secondary metabolic products.

[0082] Specifically, the farnesyl pyrophosphate synthase mentioned aboveis an enzyme involved in the metabolism of isoprenoids on which avariety of substances in plants (such as pigments, odorants,phytohormones, phytoalexins and defense substances against pests) arebased. Therefore, by using the farnesyl pyrophosphate synthase genesisolated as mentioned above, it will be possible to control the plantmetabolic systems for pigments, odorants, phytohormones, phytoalexins,and defense substances against pests and to detect the genes responsiblefor these traits.

[0083] It will also be possible to produce the secondary metabolites ofplants in vitro by using the farnesyl pyrophosphate synthase producedthrough gene manipulations using the farnesyl pyrophosphate synthasegenes isolated in this invention.

[0084] There appears to be the possibility that farnesyl pyrophosphatesynthase is involved in the metabolic systems of a hop for hop resin(hop resin constituents) and xanthohumol (Brauwelt, 36, 1998) the latterof which is said to possess anticancer action. It will further bepossible to control the metabolic systems for the hop resin andxanthohumol by using the nucleic acids of this invention as well as toutilize said nucleic acids as genetic makers for the hop resin andxanthohumol.

[0085] Accordingly, it will be possible to carry out the method ofbreeding a hop through gene manipulations that conventionally have hadto rely on experience and intuition. For example, a plant transformationtechnique is utilized to introduce the farnesyl pyrophosphate synthasegene of this invention into a hop. It will thus be possible to controlthe composition of the secondary metabolites in luplin gland.Accordingly, it will become possible to improve and maintain thequalities of foods utilizing hops (e.g., beer and low malt beer) as wellas to improve and maintain the qualities of drugs utilizing thesecondary metabolites.

[0086] BY utilizing the nucleic acid containing the promoter region ofthe farnesyl pyrophosphate synthase gene according to this invention, agene to be desirably introduced into an objective hop and a terminatorcapable of functioning in the hop are linked to the downstream of thepromoter, and this is introduced to the hop. The aforementioned genewill thus be allowed to be specifically expressed in the luplin gland.

EXAMPLES

[0087] This invention will be described more concretely by referring tothe examples; however, the invention is not to be limited by theseexamples.

Example 1 Preparation of Hop Genomic DNA

[0088] The preparation of hop genomic DNA was carried out in thefollowing manner described. Specifically, the leaves of a hop wasfreeze-ground in liquid nitrogen, suspended in a 2% CTAB solution [2%cetyltrimethylammonium bromide, 0.1 M Tris (pH 9.5), 20 mM EDTA, 1.4 MNaCl, 5% β-mercaptoethanol], and incubated at 65° C. for 30 minutes.After the suspension was extracted with chloroform/isoamyl alcohol(24:1) twice, DNA and RNA was allowed to precipitate by adding a ¾-foldamount of isopropanol. The precipitated DNA and RNA was dissolved in aHigh Salt TE buffer [1M sodium chloride, 10 mM Tris (pH 8.0), 1 mM EDTA]and it was incubated with addition of RNase at 60° C. to decompose onlyRNA. To this was added a two-fold amount of isopropanol, resulting inthe precipitation of DNA. The precipitated DNA was washed with 70%ethanol and was then dissolved to prepare a genomic DNA sample.

Example 2 Isolation of Farnesyl Pyrophosphate Synthase Gene and itsPromoter

[0089] Out of the amino acid sequences of farnesyl pyrophosphatesynthase for Arabidopsis, corn, guayule, Hevea, white lupin, and pepperwhose nucleotide sequences were known, the sequence that was consensusamong the respective plants was made a basis on which Primer 1 (SEQ IDNO:4) and Primer 2 (SEQ ID NO:5) were synthesized. These primers wereused together with the hop genomic DNA as a template to carry out PCR,producing Fragment 1 in FIG. 1.

[0090] Next, the resulting amplified fragment was sequenced. Primer 3(SEQ ID NO:6), Primer 4 (SEQ ID NO:7), Primer 5 (SEQ ID NO:8), andPrimer 6 (SEQ ID NO:9) shown in Table 1 were designed based on theobtained nucleotide sequence, and Inverse PCR was performed to obtainFragments 2 and 3 in FIG. 1. TABLE 1 SEQ ID Primer No. Nucleotidesequence 1 4 5′-GGYTGGTGYATTGAATGG-3′ 2 5 5′-TAAAAYGARTARTARGCHGTYTT-3′3 6 5′-CCTTTGGTACTCTAAACCAGCAGGG-3′ 4 7 5′-TTACAAAGTGTTAAAAGGGTATCCC-3′5 8 5′-AGGTGGAATTCCAAACAGCCTCGGG-3′ 6 9 5′-TTTGATCACCACAATTGAAGGAGAG-3′7 10 5′-GACATTGTAATCCAGCATCTGC-3′ 8 11 5′-CACAGAGAAATTGAACTTGGTC-3′ 9 125′-CACTTCCTTTGACCTGTTTG-3′ 10 13 5′-AAGCTCGTGGAGTAACCCTC-3′ 11 145′-GCGTGTTTGCGGATTACGAG-3′ 12 15 5′-TGAGAAGGATTTTGGCAGCC-3′ 13 165′-GAATTCTTATGATTAACCAAAAAC-3′ 14 175′-CGGGATCCATGAGTGGTTTAAGGTTCAAAAT-3′ 15 185′-CGGGATCCTTACTTCTGCCTCTTGTAGATC-3′

[0091] Specifically, the hop genomic DNA was digested with restrictionenzymes BglII or HindIII. A DNA Ligation Kit Ver. 1 (Takara Shuzo Co.Ltd.) was used to carry out self-ligation following the protocolattached thereto. After the self-ligation was completed, a portion ofthe reaction solution was used as a template to carry out PCR byemploying Primers 3 and 5. Subsequently, a portion of the reactionsolution for which the PCR had been completed was used as a template tocarry out PCR by employing Primers 4 and 6 shown in Table 1. Fragments 2and 3 in FIG. 1 were thus obtained.

[0092] Similarly, Primer 7 (SEQ ID NO:10), Primer 8 (SEQ ID NO:11), andPrimer 9 (SEQ ID NO:12) were designed based on the nucleotide sequenceof Fragment 2 in FIG. 1. The hop genomic DNA digested with restrictionenzyme EcoRI was subjected to self-ligation, and this was used as atemplate to carry out PCR again by employing Primers 7 and 9 describedabove. Fragment 4 in FIG. 1 was thus obtained and it was sequenced.

[0093] Fragment 5 in FIG. 1 was isolated with a Takara LA PCR in vitroCloning Kit (Takara Shuzo Co. Ltd.) by Casette-ligation mediated PCRaccording to a protocol attached thereto. Specifically, the hop genomicDNA was digested with restriction enzyme EcoRI and to this was linked anEcoRI adapter included in the kit. PCR was next carried out by usingPrimer 10 (SEQ ID NO:13) that had been designed on the basis of thenucleotide sequence of Fragment 3 and cassette primer Cl included in thekit. This PCR reaction solution was further used as a template to carryout PCR by employing Primer 11 (SEQ ID NO:14) that had been designed onthe basis of the nucleotide sequence of Fragment 3 and cassette primerC2 included in the kit. Fragment 5 was thus obtained and it wassequenced.

[0094] Finally, PCR was carried out by using the hop genomic DNA as atemplate and Primer 12 (SEQ ID NO: 15) and Primer 13 (SEQ ID NO: 16)that had been designed on the basis of Fragment 4 and Fragment 5 in FIG.1, respectively. Fragment 6 was thus obtained that contained the hopfarnesyl pyrophosphate synthase gene and a promoter thereof. All the PCRmanipulations described above were carried out using an ExpandHigh-Fidelity PCR System (Boeringer Manheim AG) according to theprotocol attached thereto.

Example 3 Sequencing of the Hop Farnesyl Pyrophosphate Synthase Gene andits Promoter

[0095] Both ends of Fragment 6 containing the hop farnesyl pyrophosphatesynthase gene and its promoter that had been obtained in Example 2 weremade blunt by using a Takara BKL Kit (Takara Shuzo Co. Ltd.) and it wassubcloned into a pUC vector. The protocol attached to the kit wasfollowed to make both ends of Fragment 6 blunt and to effect thesubcloning into the pUC vector.

[0096] Sequencing was carried out using an ABI PRISM Dye TerminatorCycle Sequencing Ready Reaction Kit (ABI373S type available from PEBiosystem Inc.) according to the protocol attached thereto. Thenucleotide sequence of Fragment 6 is shown in SEQ ID NO:2 in theSequence Listing.

Example 4 Preparation of the Respective Tissue Fractions and Total RNA

[0097] The leaves, stem, luplin (−), and luplin (+) fractions of a hopwere prepared for the tissues from which total RNA would be extracted.As used herein, “luplin (−) fraction” was a fraction principallyrecovered from the bract of a cone where luplin gland was hardlypresent. The “luplin (+) fraction” was a fraction consisting principallyof luplin gland that was obtained by removing from the cone, tissuesother than the luplin gland as much as possible. These tissue fractionswere freeze-ground in liquid nitrogen, suspended in a 2% CTAB solution[2% cetyltrimethylammonium bromide, 0.1 M Tris (pH 9.5), 20 mM EDTA, 1.4M NaCl, 5% β-mercaptoethanol], and incubated at 65° C. for 10 minutes.After the suspension was extracted with chloroform/isoamyl alcohol(24:1) twice, a ⅓-fold amount of 10M lithium chloride was added to theextract and it was allowed to stand overnight. After centrifugation at15,000 rpm for 10 minutes, the precipitates are dissolved in water. Whentotal RNA was to be used in Example 5, the precipitates were dissolvedin DNase reaction buffer [100 mM sodium acetate (pH 5.2), 5 mM magnesiumchloride] in place of water and it was incubated with addition of DNaseat 37° C. to decompose only DNA. To the solution was further added a⅓-fold amount of 10 M lithium chloride. It was allowed to standovernight and centrifuged at 15,000 rpm for 10 minutes. After washing,the precipitates were washed with 70% ethanol and were then dissolved inwater again to prepare a total RNA sample.

Example 5 Isolation and Sequencing of cDNA for the FarnesylPyrophosphate Synthase Gene

[0098] Both ends of the coding region for the hop farnesyl pyrophosphatesynthase gene were presumed that was set forth in SEQ ID NO:2 andsequenced in Example 3 based on the information about the farnesylpyrophosphate synthase gene of Arabidopsis (among others) or the likethe nucleotide sequence of which was known. Primers were designed thathad the sequences obtained by appending the BamHI recognition sequenceto those sequences and they were used with the total RNA produced inExample 4 as a template, where cDNA of the farnesyl pyrophosphatesynthase gene was isolated by RT-PCR. Specifically, Primer 14 (SEQ IDNO:17) and Primer 15 (SEQ ID NO:18) were used as primers and PCR wascarried out using a Titan One Tube RT-PCR System (Roche DiagnosticsInc.) according to the protocol attached thereto. The thus obtained cDNAof the farnesyl pyrophosphate synthase gene was subcloned into a PCR2.1vector (Invitrogen Inc.) to prepare pFPPS101R. The subcloned cDNA of thefarnesyl pyrophosphate synthase gene was sequenced using an ABI PRISMDye terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems Inc.)and a DNA sequencer ABI 373S type (PE Biosystems Inc.) according to theprotocol attached thereto. The nucleotide sequence of cDNA for theobtained farnesyl pyrophosphate synthase gene is shown in SEQ ID NO:3 inthe Sequence Listing. The amino acid sequence of the protein encoded bythis cDNA is shown in SEQ ID NO:1.

[0099] Within the nucleotide sequence set forth in SEQ ID NO:3, the660th base differed from the corresponding base (the 3737th base in SEQID NO:2) in the genomic DNA, although confirmation of the nucleotidesequences was conducted plural times. This is thought to be a baseincorporation error resulting from RT-PCR conducted when cDNA wasisolated. However, this error does not affect the functional analysis ofprotein as will be described in Example 6 because it leads to the aminoacid identical to that which should have been encoded at the amino acidlevel.

Example 6 Functional Analysis of the Protein Encoded by the IsolatedFarnesyl Pyrophosphate Synthase Gene

[0100] In order to determine whether or not the protein encoded by theisolated farnesyl pyrophosphate synthase gene possessed the farnesylpyrophosphate synthase activity, cDNA of the farnesyl pyrophosphatesynthase gene isolated in Example 5 was treated with restriction enzymeBamHI to give DNA. The DNA was incorporated into the BamHI site of anexpression vector pQE30 [attached to a QIAexpress Expression System(QIAGEN Inc.)]. It was then introduced into E. coli to have the farnesylpyrophosphate synthase gene expressed in the E. coli cells and theexpressed product was purified. Expression of the farnesyl pyrophosphatesynthase gene in the E. coli cells and purification of the expressedproduct was carried out according to the protocol attached to aQIAexpress Expression System (QIAGEN Inc.).

[0101] Next, the method of Sylvie A. et al. (Arch. Biochem. Biophys.321, 493-500 (1995)) was followed to determine whether or not theobtained expression product possessed the farnesyl pyrophosphatesynthase activity. Specifically, to 100 μl of enzyme reaction solution(50 mM Tris-HCl, 2 mM dithioerythritol, 1 mM magnesium chloride, 100 μMdimethylallyl pyrophosphate) were added 2 μl (28 μg) of the purifiedexpression product of the farnesyl pyrophosphate synthase gene and 2.5μl (0.05 μCi) of ¹⁴C-isopentenyl pyrophosphate, and the reaction wasallowed to take place at 30° C. for 30 minutes. To alkaline phosphatase(Wako Pure Chemical Industries, Ltd.) was added 30 μl of 10-foldconcentration reaction buffer (as attached). Alkaline phosphatase, 1 μl,(10 units) was then added to the reaction, which was allowed to continueat 37° C. for 3 hours. Then, the reaction was further continued at 25°C. overnight. To the reaction solution was added 1 μl of farnesol (4.5nmol) as a carrier and further 200 μl of hexane, and upon mixing thehexane layer was recovered after centrifugation at 10,000 rpm for oneminute. Hexane, 100 μl, was again added to the remaining water layer andit was mixed and centrifuged to recover a hexane layer, which wascombined with the hexane layer recovered earlier. The hexane extract wasconcentrated to 1 μl by being blown with nitrogen gas. After 10 μl ofmethanol was added and mixed to the concentrate, 1 μl was spotted ontothin layer chromatography (HPTLC-aluminum sheets silica gel 60 F254pre-coated available from Merk KGaA) and developed in a developingsolvent (benzene:ethyl acetate=9:1). Farnesol and geraniol weresimultaneously spotted as a standard. After the thin layer chromatogramplate for which development had been completed was dried, iodine wassprayed onto the plate to ascertain the positions of farnesol andgeraniol. Exposure to a X-ray film was carried out at −80° C. for 7days. The obtained results are shown in FIG. 4.

[0102] As FIG. 4 shows, the signals of the reaction products weredetected at the positions of farnesol and geraniol. Specifically, it wasconfirmed that the protein encoded by the isolated farnesylpyrophosphate synthase gene possessed the farnesyl pyrophosphatesynthase activity.

Example 7 Northern Hybridization

[0103] Northern hybridization was carried out to determine in whichtissue of a hop the isolated farnesyl pyrophosphate synthase gene wasexpressed as well as to determine the level of its expression.

[0104] Plasmid pFPPS101R prepared in Example 5 was first digested withrestriction enzyme KpnI to produce a linearized form. A DIG RNA labelingkit (SP6/T7) (Roche Diagnostics Inc.) was used to prepare a RNA probefor the farnesyl pyrophosphate synthase gene by employing the linearizedform as a template. The preparation method was performed according tothe protocol attached to the kit.

[0105] The total RNAs for the leaves, the stem, the luplin (−) fraction,and the luplin (+) fraction that had been prepared in Example 4 (each 15μl) were subjected to electrophoresis using denatured agarose gel (1.2%agarose, 6.7% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA,pH 7.0). The gel for which electrophoresis had been completed was shakenin distilled water three times each for 40 minutes and afterformaldehyde in the agarose gel was removed, RNA in the agarose gel wastransferred to a nylon membrane by using 20×SSC (0.3 M sodium citrate, 3M sodium chloride, pH 7.0) as buffer. The nylon membrane to which RNAhad been transferred and the aforementioned probe were used to carry outhybridization at 68° C. overnight. Here, the composition of thehybridization buffer used in the hybridization was 5×SSC, 0.02% SDS,0.1% N-lauroylsarcosin, 50% formamide, and 2% Blocking Reagent (RocheDiagnostics Inc.). After hybridization, a detergent (0.1% SDS, 2×SSC)was used to carry out washing-treatment twice each at 68° C. for 30minutes; further, a detergent (0.1% SDS, 0.1×SSC) was used to carry outwashing-treatment twice each at 68° C. for 30 minutes. After washing,the RNA fragment to which the probe had been hybridized was detected.The detection was carried out according to the protocol described in“The DIG System User's Guide for Filter Hybridization” (Roche DiagnosticInc.). The results obtained are shown in FIG. 5.

[0106] From the results of FIG. 5, there was observed in each of thetissue fractions, the leave, the stem, the luplin (−) fraction, and theluplin (+) fractions a signal at the position of 1.1 kb approximating tothe size of the nucleic acid having the nucleotide sequence set forth inSEQ ID NO:2 in the Sequence Listing that encodes the farnesylpyrophosphate synthase gene. This confirmed that the hop farnesylpyrophosphate synthase gene was expressed in each of the tissuefractions. However, the intensity of the signal was in the order of theluplin (+) fraction >the luplin (−) fraction >the stem >the leaf. Thisconfirmed that the degree of mRNA derived from the farnesylpyrophosphate synthase gene occupying in each tissue was the greatest inthe luplin gland, next place in the stem and the bract, and the least inthe leaf. In other words, it was confirmed that the farnesylpyrophosphate synthase gene was expressed in the strongest manner in theluplin gland and thus the promoter of the farnesyl pyrophosphatesynthase gene had the strongest promoter activity in the luplin gland.

INDUSTRIAL APPLICABILITY

[0107] As described above, it is possible to identify the farnesylpyrophosphate synthase proteins and genes according to this invention.It will, therefore, reveal the genes involved in the biosynthesis ofsecondary metabolites in a hop as well as the nucleotide sequences ofthe promoter genes that function in the luplin gland of the hop in atissue-specific manner. This will allow for the transformation of thehop by gene manipulations and the in vitro synthesis of the hopsecondary metabolites.

1 18 1 1029 DNA Humulus lupulus L. CDS (1)..(1029) 1 atg agt ggt tta aggtca aaa ttc atg gag gtt tac tcc att ttg aaa 48 Met Ser Gly Leu Arg SerLys Phe Met Glu Val Tyr Ser Ile Leu Lys 1 5 10 15 tca gag ctt ctc aacgat cct gct ttc gag ttc acc gat gat tct cgc 96 Ser Glu Leu Leu Asn AspPro Ala Phe Glu Phe Thr Asp Asp Ser Arg 20 25 30 caa tgg gtc gaa cgg atgctg gat tac aat gtc cca gga ggt aag ctt 144 Gln Trp Val Glu Arg Met LeuAsp Tyr Asn Val Pro Gly Gly Lys Leu 35 40 45 aat cgt ggg ttg tca gtt attgat agt tac caa tta ctt aaa gga gga 192 Asn Arg Gly Leu Ser Val Ile AspSer Tyr Gln Leu Leu Lys Gly Gly 50 55 60 aag gaa cta act gaa gaa gaa attttt cta act tct gct ctt ggt tgg 240 Lys Glu Leu Thr Glu Glu Glu Ile PheLeu Thr Ser Ala Leu Gly Trp 65 70 75 80 tgt att gaa tgg ctt caa gca tatttt ttg gtt ctt gat gat atc atg 288 Cys Ile Glu Trp Leu Gln Ala Tyr PheLeu Val Leu Asp Asp Ile Met 85 90 95 gat aac tct gtt aca cgt cgc ggt caaccc tgc tgg ttt aga gta cca 336 Asp Asn Ser Val Thr Arg Arg Gly Gln ProCys Trp Phe Arg Val Pro 100 105 110 aag gtt gga ttg att gct gca aac gatggc att cta ctt cga aac cat 384 Lys Val Gly Leu Ile Ala Ala Asn Asp GlyIle Leu Leu Arg Asn His 115 120 125 att ccg aga att ctt aag aag cat ttcaag ggg aag agc tac tat gtg 432 Ile Pro Arg Ile Leu Lys Lys His Phe LysGly Lys Ser Tyr Tyr Val 130 135 140 gat ctt ctt gat ttg ttt aat gag gtggaa ttc caa aca gcc tcg gga 480 Asp Leu Leu Asp Leu Phe Asn Glu Val GluPhe Gln Thr Ala Ser Gly 145 150 155 160 caa atg att gat ttg atc acc acaatt gaa gga gag aaa gat ctt tca 528 Gln Met Ile Asp Leu Ile Thr Thr IleGlu Gly Glu Lys Asp Leu Ser 165 170 175 aaa tac tca att cca ctt cac catcgc att gtt cag tac aag act gct 576 Lys Tyr Ser Ile Pro Leu His His ArgIle Val Gln Tyr Lys Thr Ala 180 185 190 tac tac tca ttc tac ctt ccg gttgct tgt gca ttg gtg atg gct ggt 624 Tyr Tyr Ser Phe Tyr Leu Pro Val AlaCys Ala Leu Val Met Ala Gly 195 200 205 gaa aat ctt gat aac cat gtt gatgtg aag aac gtc ctt att gaa atg 672 Glu Asn Leu Asp Asn His Val Asp ValLys Asn Val Leu Ile Glu Met 210 215 220 gga acc tat ttc caa gta cag gatgac tat ttg gat tgt ttt ggc cac 720 Gly Thr Tyr Phe Gln Val Gln Asp AspTyr Leu Asp Cys Phe Gly His 225 230 235 240 cca gat gta att ggc aag attggt aca gat att gaa gac ttc aag tgc 768 Pro Asp Val Ile Gly Lys Ile GlyThr Asp Ile Glu Asp Phe Lys Cys 245 250 255 tct tgg ttg gtt gtg aaa gcactc gaa ata gct acc gag gaa caa aag 816 Ser Trp Leu Val Val Lys Ala LeuGlu Ile Ala Thr Glu Glu Gln Lys 260 265 270 aag atg cta ttt gag cat tatggt aag gga gat gaa gca tct gtc aaa 864 Lys Met Leu Phe Glu His Tyr GlyLys Gly Asp Glu Ala Ser Val Lys 275 280 285 aaa gtg aaa gag tta tac aaggcc ctt gat ctt gag ggc gtg ttt gcg 912 Lys Val Lys Glu Leu Tyr Lys AlaLeu Asp Leu Glu Gly Val Phe Ala 290 295 300 gat tac gag aat gct agt taccaa aag ctt ata aaa tcg atc gaa gct 960 Asp Tyr Glu Asn Ala Ser Tyr GlnLys Leu Ile Lys Ser Ile Glu Ala 305 310 315 320 cat ccg aaa gaa gaa gttcag gca gtg ctc aaa tct ttc ttg gct aag 1008 His Pro Lys Glu Glu Val GlnAla Val Leu Lys Ser Phe Leu Ala Lys 325 330 335 atc tac aag agg cag aagtaa 1029 Ile Tyr Lys Arg Gln Lys 340 2 342 PRT Humulus lupulus L. 2 MetSer Gly Leu Arg Ser Lys Phe Met Glu Val Tyr Ser Ile Leu Lys 1 5 10 15Ser Glu Leu Leu Asn Asp Pro Ala Phe Glu Phe Thr Asp Asp Ser Arg 20 25 30Gln Trp Val Glu Arg Met Leu Asp Tyr Asn Val Pro Gly Gly Lys Leu 35 40 45Asn Arg Gly Leu Ser Val Ile Asp Ser Tyr Gln Leu Leu Lys Gly Gly 50 55 60Lys Glu Leu Thr Glu Glu Glu Ile Phe Leu Thr Ser Ala Leu Gly Trp 65 70 7580 Cys Ile Glu Trp Leu Gln Ala Tyr Phe Leu Val Leu Asp Asp Ile Met 85 9095 Asp Asn Ser Val Thr Arg Arg Gly Gln Pro Cys Trp Phe Arg Val Pro 100105 110 Lys Val Gly Leu Ile Ala Ala Asn Asp Gly Ile Leu Leu Arg Asn His115 120 125 Ile Pro Arg Ile Leu Lys Lys His Phe Lys Gly Lys Ser Tyr TyrVal 130 135 140 Asp Leu Leu Asp Leu Phe Asn Glu Val Glu Phe Gln Thr AlaSer Gly 145 150 155 160 Gln Met Ile Asp Leu Ile Thr Thr Ile Glu Gly GluLys Asp Leu Ser 165 170 175 Lys Tyr Ser Ile Pro Leu His His Arg Ile ValGln Tyr Lys Thr Ala 180 185 190 Tyr Tyr Ser Phe Tyr Leu Pro Val Ala CysAla Leu Val Met Ala Gly 195 200 205 Glu Asn Leu Asp Asn His Val Asp ValLys Asn Val Leu Ile Glu Met 210 215 220 Gly Thr Tyr Phe Gln Val Gln AspAsp Tyr Leu Asp Cys Phe Gly His 225 230 235 240 Pro Asp Val Ile Gly LysIle Gly Thr Asp Ile Glu Asp Phe Lys Cys 245 250 255 Ser Trp Leu Val ValLys Ala Leu Glu Ile Ala Thr Glu Glu Gln Lys 260 265 270 Lys Met Leu PheGlu His Tyr Gly Lys Gly Asp Glu Ala Ser Val Lys 275 280 285 Lys Val LysGlu Leu Tyr Lys Ala Leu Asp Leu Glu Gly Val Phe Ala 290 295 300 Asp TyrGlu Asn Ala Ser Tyr Gln Lys Leu Ile Lys Ser Ile Glu Ala 305 310 315 320His Pro Lys Glu Glu Val Gln Ala Val Leu Lys Ser Phe Leu Ala Lys 325 330335 Ile Tyr Lys Arg Gln Lys 340 3 4699 DNA Humulus lupulus L. 3tgagaaggat tttggcagcc tcttaaattt gacaaaattt tgattggtta tatatatata 60tatgtttaca tttattgtgt atgcttgcca aacaacatat gatgatatat tacttttttg 120tcctcactct ttgattttgt tttttattat tctaaacttt gtaattttcc attcaagctt 180acatttctat catatattat aatattattt ttgtcatatt aattatatca aaatacaaaa 240aattaacatt ttagtctatt ttacaaaatc tactaaaatt aatttccatt ttgatattaa 300aaaaataaaa cattcatata aaattgacca aaagctaatg tatggttaat tttgataaat 360aatgtaaaac attatataat tactaagttt tcttaaatta acataaagtc taatatttag 420ctaaaatcaa ataatatatt tcttatataa aagcaataaa aaaaagtggt cttgtatgat 480tattcaattt tttttctttg tttaattttt ctatgtcata catattataa caataataag 540attcttttac ataacatgaa ttttttccat ttgagtttag atgagtctac tttgctcttt 600aaaggaaaaa ttataacaat aacatgagtt tttttttttt catttaagtt ttttttgctc 660cttaatagta aatattagtt aatatatata attttaaaaa aatatttttt tttaaattaa 720tctattcaac cactaaattt cattttagta gtcaatctca tacaaataca aacattataa 780ttacacataa atcattactc taataatata taataaaata aagaagagtt tttttcttaa 840ttaaaattat tttgttttca cacattataa caataaataa tgagaggttc ttacatatga 900aactcattta atttaacaat atttgtctat catgctcttt aaataaaaaa taacaacaat 960aacaattatt atattatata tatattatat aggacaattc tctttatacc ctaccgaata 1020gtttacagta ccaaaaatta ggttaaaaaa tattgttttc tacgcacata aaaaaaaaat 1080taatcacggg tgtaacaaca tgtttgaatt tagttttcgt tactgtaaac tattcagaat 1140tttatgaaaa tttgcagatg ctctaaataa ctacaatata tacggtcata aaaaaaatcg 1200cgccgaaaac tgttcacaag tcaagaacac tgagagtctc atcggtaaag cttaaattgt 1260agccctataa aagaattatc ctataaatat atattgtatt tatacatttt tagactctgt 1320gttttgacaa attatttttt ggaccctata ttttgtaaaa tagttcaaat aagcccttaa 1380tcctgatttt gatgaacaaa aaattaagta taacaagaca gttcttatgt agaatgatta 1440tatttttgtt ctgagttgtt agtttgatga attatttgtt atttttgttc aaaaaacttt 1500gatcaaaatc gagtttaggg gtctatttga actattttag aaaacacaag gtctaaaaaa 1560taatttatca aaacacaggt ccaaacaagt aatgagacaa aatacagaat tttaaaaaat 1620ataaacccat atatatatat atatttaaaa aatggcaaat ttgtaattat atgataaatg 1680aagccctaat gttgatgatt taaacaatgc atgattggtc aaaacaagtg gcctccaaag 1740ctctataaag agagcacttc tacactgact ctctctctct atctgtctct atatatatat 1800atttatatat agattttcag tatcacagac cagaacaagt acaacccatt agtcccacct 1860ctgttgagct cttgttcagc caaaaaatga gtggtttaag gtcaaaattc atggaggttt 1920actccatttt gaaatcagag cttctcaacg atcctgcttt cgagttcacc gatgattctc 1980gccaatgggt cgaacgggta ttcccatctc tctaactctc ttcttttttc actgacattg 2040ctttcgtttt ctccatttta tggaatttgg gttctgattt gtgtagttct caccagattc 2100gttttggtct tttcaattca aactattttt ttatatgtaa aagtggaaac tagaacatat 2160ttccttaaaa gatctggact tttagctgaa aattttggtg ggttataatt attttcttta 2220aattagttat attatgctca gattttatct gatcttgttc attgaagaat cctcttcacc 2280atgtttaaat actgtgttat tattatcgta ttctctcttt agttttttca ttaagattct 2340ctattgtact gacttgtacc ttattaaaaa aacctgattg agtgtcatta tacttatttt 2400ctttcaatta ctgagactat gctcagattt tatatgatct tgttcattga agagtcatgc 2460ttagaccaag ttcaatttct ctgtgtagtt ctttctttaa gattctttat tgtacttagt 2520tgagccttat aaaaagacct gattgagtgt cattttttac tttgcagatg ctggattaca 2580atgtcccagg aggtttggaa tgcaactctt ttttcacttc ctttgacctg tttgtgttga 2640gaatgattgt aatgaactgt gacattttac cttctttaac aaattcatat tgattttttc 2700tggttggtag gtaagcttaa tcgtgggttg tcagttattg atagttacca attacttaaa 2760ggaggaaagg aactaactga agaagaaatt tttctaactt ctgctcttgg ttggtgtatt 2820gaatgggtat gcagctttca ttttggtact ttttattcat taattgggat acccttttaa 2880cactttgtaa cattcaattc tatgtagctt caagcatatt ttttggttct tgatgatatc 2940atggataact ctgttacacg tcgcggtcaa ccctgctggt ttagagtacc aaaggtgtga 3000ctttacatcc tttcatgggg tttttctctt gttttctatg gagtgaatat cctaacatgt 3060gtaagtgttc tgcgtttatc acaggttgga ttgattgctg caaacgatgg cattctactt 3120cgaaaccata ttccgagaat tcttaagaag catttcaagg ggaagagcta ctatgtggat 3180cttcttgatt tgtttaatga ggtgtgattt gtttgatgga tagttgagta gagcaaaagg 3240gtttcttttt ctttgtcatc ttagtcatgt aacagggttt gttaatctac aggtggaatt 3300ccaaacagcc tcgggacaaa tgattgattt gatcaccaca attgaaggag agaaagatct 3360ttcaaaatac tcaattccac tgtaagtgaa attgatatgt gtgattacta caagactttc 3420actcaaatat tcgatgtgcc taaagattgt ggtgttgtct tgcagtcacc atcgcattgt 3480tcagtacaag actgcttact actcattcta ccttccggta agagagaacc gttgatttat 3540gcattcatga agcatgttgc cttgattggt tttcatcttt gtctttgata gttctgcttg 3600ttttgataat ctttttcatg taagaatcta tcaactgcgc ttatgtatcg ttctctgaca 3660ctaattatga tcaggttgct tgtgcattgg tgatggctgg tgaaaatctt gataaccatg 3720ttgatgtgaa gaacgttctt attgaaatgg gaacctattt ccaagtacag gtgattgcag 3780acatatcctc atttgtatat tcttgagaaa tatagttgca aatgtattga ccaaatcttt 3840tggccactga tcctgataat cttgctgaac attttactgt atttttattg acaaaaaaac 3900caatgacata ttatgtagga tgactatttg gattgttttg gccacccaga tgtaattggc 3960aaggtatgtt ataaagccac actcttttgt caagtttgta atgcactatc ctcattcaac 4020tgggaatttt tttcagtttt cattgatgcc cttttggatt atcagattgg tacagatatt 4080gaagacttca agtgctcttg gttggttgtg aaagcactcg aaatagctac cgaggaacaa 4140aagaagatgc tatttgtaag caataaagtt catcctttca atttttacat gaaaatgtct 4200cgagggaacg ttggttactt ttgcttacac atctgaattc tgtgttttcc aggagcatta 4260tggtaaggga gatgaagcat ctgtcaaaaa agtgaaagag ttatacaagg cccttgatct 4320tgaggttcat tctctctgtt tctctttcga tctatgtttt taaagctcgt ggagtaaccc 4380tcttctaact tcgatttttg ggatttgtag ggcgtgtttg cggattacga gaatgctagt 4440taccaaaagc ttataaaatc gatcgaagct catccgaaag aagaagttca ggcagtgctc 4500aaatctttct tggctaagat ctacaagagg cagaagtaag gttgaaaatg gagatttgga 4560ctaaagagat aacaatcaac tttgtgtggg cattagcatt tctttcactc tttttaataa 4620aagggtcatt tttagtgatt gtttttggtt aatcataaga attcttagtt catcttatgc 4680tgagtggtgg atattttaa 4699 4 18 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 4ggytggtgya ttgaatgg 18 5 23 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 5taaaaygart artargchgt ytt 23 6 25 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA6 cctttggtac tctaaaccag caggg 25 7 25 DNA ARTIFICIAL SEQUENCE SYNTHETICDNA 7 ttacaaagtg ttaaaagggt atccc 25 8 25 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 8 aggtggaatt ccaaacagcc tcggg 25 9 25 DNA ARTIFICIALSEQUENCE SYNTHETIC DNA 9 tttgatcacc acaattgaag gagag 25 10 22 DNAARTIFICIAL SEQUENCE SYNTHETIC DNA 10 gacattgtaa tccagcatct gc 22 11 22DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 11 cacagagaaa ttgaacttgg tc 22 1220 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 12 cacttccttt gacctgtttg 20 1320 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 13 aagctcgtgg agtaaccctc 20 1420 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 14 gcgtgtttgc ggattacgag 20 1520 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 15 tgagaaggat tttggcagcc 20 1624 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 16 gaattcttat gattaaccaa aaac24 17 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 17 cgggatccat gagtggtttaaggtcaaaat 30 18 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 18 cgggatccttacttctgcct cttgtagatc 30

1. A protein having the amino acid sequence set forth in SEQ ID NO:1 inthe Sequence Listing.
 2. A protein having an amino acid sequencederivable from the deletion or the substitution of one or more aminoacids in the amino acid sequence set forth in SEQ ID NO:1 in theSequence Listing, or from the addition of one or more amino acids to theamino acid sequence set forth in SEQ ID NO:1 in the Sequence Listing,said protein possessing the farnesyl pyrophosphate synthase activity. 3.A nucleic acid encoding a protein having the amino acid sequence setforth in SEQ ID NO:1 in the Sequence Listing.
 4. A nucleic acid havingthe nucleotide sequence set forth in SEQ ID NO:3 in the SequenceListing.
 5. A nucleic acid comprising a part of the nucleotide sequenceset forth in SEQ ID NO:3 in the Sequence Listing.
 6. A nucleic acid thathybridizes to a nucleic acid according to claim 4 or 5, or to acomplementary nucleic acid thereof under stringent conditions, saidnucleic acid encoding a protein possessing the farnesyl pyrophosphatesynthase activity.
 7. A nucleic acid having the nucleotide sequence setforth in SEQ ID NO:2 in the Sequence Listing.
 8. A nucleic acidcomprising a part of the nucleotide sequence set forth in SEQ ID NO:2 inthe Sequence Listing.
 9. A nucleic acid having a nucleotide sequence offrom base No. 1 to base No. 1886 in the nucleotide sequence set forth inSEQ ID NO:2 in the Sequence Listing.
 10. A nucleic acid that hybridizesto a nucleic acid according to any of claims 7-9 or to a complementarynucleic acid thereof under stringent conditions, said nucleic acidpossessing promoter activity.